One goal in the DNA sequencing industry is to reduce the cost and increase the speed for sequencing an individual's genome. A target accuracy of 99.99%1 is also important. Another vital goal is long contiguous reads. Array technology is revealing a remarkable long range complexity in the genome, some of which is summarized in Table 1 below, taken from the review by Sharp et al.2. This complexity is difficult to retrieve through the assembly of contigs much shorter than the size ranges referred to in the table. Copy number variations are particularly vexing, yet they have important phenotypes owing to gene-dose variations. These clinically-important copy number variations are almost completely uncorrelated with the SNPs extracted by conventional re-sequencing.3
TABLE 1Long-range structures in the genome (from Sharpeet al. Ann. Rev. Hum. Gen. 17 407 2006)VariationRearrangement typeSize rangeaSingle base-pair changesSingle nucleotide polymorphisms, point1bpmutationsSmall insertions/deletionsBinary insertion/deletion events of short1-50bpsequences (majority < 10 bp in size)Short tandem repeatsMicrosatellites and other simple repeats1-500bpFine-scale structural variationDeletions, duplications, tandem repeats,50 bp to 5 kbinversionsRetroelement insertionsSINEs, LINEs, LTRs, ERVsb300 bp to 10 kbIntermediate-scale structuralDeletions duplications, tandem repeats,5 kb to 50 kbvariationinversionsLarge-scale structural variationDeletions, duplications, large tandem repeats,50 kb to 5 MbinversionsChromosomal variationEuchromatic variants, large cytogenetically~5 Mb to entirevisible deletions, duplications, translocations,chromosomesinversions, aneuploidy
Sequencing by extension relies on detection of a signal owing to nucleotide addition by a polymerase and instruments using many molecules in picoliter wells are already in commercial use.4 For example, the 454 Life Sciences instrument exploits energetic pyrophosphates to give label-free optical signals, but the length of the read is limited by the dephasing problem as individual clones in a well fall behind owing to errors in nucleotide addition. Single molecule measurements present a way around this problem5,6 and this is the basis of the Helicos system. However, the chemistry required to do this is far from trivial and the use of non-natural nucleotides limits the length of the extensions. An interesting variant is the proposal to sequence by ligation (Xiaohua lab, UCSD7).
The use of the scanning tunneling microscope (STM) for sequencing DNA (AFM and STM in novel approaches to sequencing) has been studied in the past. DNA has been imaged at high resolution in water8-10 but sequencing was not possible. In part, this is because the magnitude of the tunneling current through DNA in terms of base composition has not been easy to interpret. In addition, the STM images themselves are not amenable to ready interpretation. These images often reflect where the structure makes contact with the underlying metal and not the real high points of the molecule.11 This complication may also affect the recent STM imaging work of Oshiro and Umezawa.12 This difficulty rules out sequencing by imaging.
The nanopore approach13-25 has the great advantage of only allowing one base to pass a particular point at a time (if the orifice is small enough). It can also be highly precessive (moving from one base to the next without “stuttering”) if the driving force is high enough. For instance, if one were to assume that an allowable limit on stuttering misreads of 1 part in 104 (99.99% accuracy), this would require a driving free-energy of 9.3 kBT. To achieve this requires either a voltage drop across a base of 0.23V (a field of 4×108V/m) or a force of 55 pN (where a base-to-base separation of 0.6 nm for stretched DNA was used26). These conditions are readily achieved and demonstrable.
Since Kasianowicz13 employed a biological nanopore, the alpha-hemolysin ion channel, as a sensor to characterize DNA, nanopore based biosensors have attracted much attention.21,27,28 However, biological nanopores have several shortcomings as they are unstable, fragile and fixed in size. Synthetic single nanopores29-33 were developed as alternatives to biological nanopores. These artificial single nanopores have been successfully used to characterize DNA translocation, folding, and conformational changes,34-37 the effects of high pH,38 low temperatures,39 multiple DNA lengths,40 the effects of electric field strength,41 the effects of surface modification by atomic layer deposition (ALD)42 and also for mimicking ion channel activity.43 Solid-state single nanopores now have a real track record. They have the following advantages: (1) the pore size is comparable to that of a single molecule; (2) the pore size is tunable to fit a wide range of molecules; and (3) robust single channels between cis and trans reservoirs are readily achieved. They also have the following strengths as a manufacturable device: (1) they are chemically and thermally stable; (2) their surfaces are readily modified; (3) they are mechanically robust; and (4) they are readily incorporated into integrated circuit (IC) technology.
It was hoped originally that the DNA bases could be identified directly via distinctive variations in the ionic current through the nanopore as each base occluded the pore on transit of the smallest part of the pore. However, the ionic current signal has proved difficult to interpret. As a result, several new schemes have been proposed. One method relies on electronic measurement of tunnel current as the DNA bases pass through a tiny gap between a pair of electrodes44-46 though there is some debate about the feasibility of this approach.47 Another proposes to exploit the distinctive dipoles of the bases with measurement of dielectric response on the molecular scale.48 Yet another proposes to measure the optical response as dye-labeled complimentary strands are “peeled off” the main template strand by passage through the nanopore.49 A group associated with the present inventors has focused on measuring the force associated with translocation.50 At the time of writing, there are no published reports of a signal with single-base resolution.49 
FIG. 2 shows the layout of a proposed sequence reader that relies on differences in the tunnel-transport of electrons through bases (taken from a recent review by Zwolak and Di Ventra49). To understand the obstacles laying in the way of operating a reader such as this one, it is useful to take a look at the history of single molecule electronic measurements. When it comes to the conductance of a molecule placed between metal electrodes, published experimental data are all over the map51 and DNA is a wonderful illustration of the problem. DNA has been reported to be an insulator,52 semiconductor,53 conductor54 and even superconductor55 (though the issue is now probably resolved56). With the exception of one datum52 these results are for conduction along the strand, but the problems inherent in making these measurements are the same no matter what the geometry is. Some of these problems include:
(1) Tunnel transport is exponentially sensitive to the atomic arrangement of atoms in the tunneling path and even a bond rotation can change transport by a significant amount.57,58 
(2) Outside of ultrahigh vacuum, metal surfaces are covered with (unknown amounts of) adventitious contamination leading to dramatic variations in contact.59 
(3) Base sequence, fluctuations of structure, and very importantly, counter ions, can dominate the electronic properties of a polyelectrolyte like DNA.60 
FIG. 3 shows an experimental arrangement that captures some of the elements that would be required for any electronic sequencing of DNA. The experimental arrangement permits reproducible determination of single molecule conductance, albeit practicing the art on a rather simple octane-dithiol molecule.61 The target molecule was chemically-contacted (to a planar bottom electrode and a nanoparticle top electrode) using gold-thiol chemistry to form a metal-chemical bond-molecule-chemical bond-metal sandwich. The resulting reproducible data showed, very clearly, the effects of having different numbers of molecules in the gap. The experiment works because the tip is not well-connected to the surrounding monolayer of octane monothiols, at best touching the terminal methyl groups, but most likely interacting via a layer of contaminant molecules. But the contamination is displaced by the chemical bonding in the case of the desired thiolated top-contact. FIG. 3A shows a single dithiolated octane molecule 31 inserted into a defect in a monolayer 32 of octane-monothiol molecules on a gold electrode 33 and a gold nanoparticle 34 is chemically attached as a top electrode. A metal-coated AFM probe 35 makes contact with the nanoparticle 34 to complete the circuit. As seen in FIG. 3B, different contacts produce different current-voltage curves 36a-e, but these are all integral multiples of each other, interpreted as integer numbers of molecules (1, 2, 3 . . . ) in the gap. FIG. 3C shows the superposition 37 that occurs when each curve is divided at all points by the appropriate integer. FIG. 3D shows the effective multiplier for thousand of curves—over 1000 contacts fell in the ‘single molecule’ bin.
FIG. 4 shows that the conductance through the desired path 44 is over a thousand times higher than the conductance through the non-bonded path 46. The conductance measured for a single molecule was quite close to what was predicted by a first principles theory 42 with no adjustable parameters61 (and the remaining small discrepancy is now explained62). This is to be contrasted with a “best case” agreement of a factor of 500 achieved in previous reports of single molecule electronic properties.51 Chemical bonds, in and of themselves, only enhance tunnel current by a few times.63 The most important factor is probably the role that bonding plays in displacing contamination from the tunnel junction, as metal surfaces are invariably coated with hydrocarbons outside of an ultra-clean, ultrahigh vacuum environment.59 Subsequent to the above-described experimental arrangements for measurement of single molecule conductance, a variety of measurements and techniques have evolved.34,51,57,58,61,64-82 83-86 In addition, there have been very significant contributions to the methods from the Tao group77 and the Columbia group.87,88 Many issues remain to be resolved, and it is important to point out that, even with the best current methods, different atomic arrangements of the contact at the electrode can lead to differences in the measured conductance.85,86,89 
Hydrogen-bond mediated STM image-enhancement has been reported.12 In addition, it is now known that one may directly measure hydrogen bond enhanced tunneling. Therefore, one area of study is to build an electrical readout system that incorporates a pair of electrodes in a nanopore. Nanopores with electrical contacts are being constructed by several groups pursuing sequencing by tunneling44-46 or capacitance measurement.48 An electrical readout would be difficult with biomolecular nanopores13 and almost certainly requires the use of a solid state nanopore. Registration of a pair of electrodes with a small (about 2-3 nm diameter) nanopore is not an easy task. Little material exists in the literature, but the two leading groups working in this area are the Harvard Nanopore Sequencing Group, where Golovchenko leads an effort towards solid state nanopore sequencing29 and Timp's group at UIUC, which is pursuing dielectric nanopore sequencing.48 The Harvard group is using a carbon nanotube placed across the nanopore that is judiciously cut so that a nm-scale gap in the electrodes lies just above the pore. The Timp group is working to build a layered semiconductor capacitor, with conductive elements separated by a sub-nm insulating spacer.48,49 The DNA would pass through a nanopore drilled perpendicular to these layers. However, sequencing of translocating DNA through such pores has proven to be elusive.